Complete Conservation Research Articles

Dearomative Intramolecular (4+3) Cycloadditions of Thiophenes

AbstractUnexpectedly facile dearomative intramolecular (4+3) cycloadditions of thiophenes with epoxy enolsilanes, providing sulfur‐bridged cycloadducts, are reported. A total of fifteen thiophene substrates have been found to undergo (4+3) cycloaddition smoothly to produce endo and exo (4+3) adducts in yields of up to 83 % with moderate to good diastereoselectivity. Complete conservation of enantiomeric purity was observed when the optically enriched epoxide was used. The desulfurizing transformations of the sulfur‐bridged skeleton of the cycloadducts provide functionalized 6,7‐fused bicyclic frameworks consisting of 1,3‐cycloheptadiene subunits. Density functional theory calculations reveal the origins of the facile dearomatization of thiophenes in these (4+3) cycloadditions.

Dearomative Intramolecular (4+3) Cycloadditions of Thiophenes.

Unexpectedly facile dearomative intramolecular (4+3) cycloadditions of thiophenes with epoxy enolsilanes, providing sulfur-bridged cycloadducts, are reported. A total of fifteen thiophene substrates have been found to undergo (4+3) cycloaddition smoothly to produce endo and exo (4+3) adducts in yields of up to 83 % with moderate to good diastereoselectivity. Complete conservation of enantiomeric purity was observed when the optically enriched epoxide was used. The desulfurizing transformations of the sulfur-bridged skeleton of the cycloadducts provide functionalized 6,7-fused bicyclic frameworks consisting of 1,3-cycloheptadiene subunits. Density functional theory calculations reveal the origins of the facile dearomatization of thiophenes in these (4+3) cycloadditions.

Sequencing of Kaposi's Sarcoma Herpesvirus (KSHV) genomes from persons of diverse ethnicities and provenances with KSHV-associated diseases demonstrate multiple infections, novel polymorphisms, and low intra-host variance.

Recently published near full-length KSHV genomes from a Cameroon Kaposi sarcoma case-control study showed strong evidence of viral recombination and mixed infections, but no sequence variations associated with disease. Using the same methodology, an additional 102 KSHV genomes from 76 individuals with KSHV-associated diseases have been sequenced. Diagnoses comprise all KSHV-associated diseases (KAD): Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated large cell lymphoma (KSHV-LCL), a type of multicentric Castleman disease (KSHV-MCD), and KSHV inflammatory cytokine syndrome (KICS). Participants originated from 22 different countries, providing the opportunity to obtain new near full-length sequences of a wide diversity of KSHV genomes. These include near full-length sequence of genomes with KSHV K1 subtypes A, B, C, and F as well as subtype E, for which no full sequence was previously available. High levels of recombination were observed. Fourteen individuals (18%) showed evidence of infection with multiple KSHV variants (from two to four unique genomes). Twenty-six comparisons of sequences, obtained from various sampling sites including PBMC, tissue biopsies, oral fluids, and effusions in the same participants, identified near complete genome conservation between different biological compartments. Polymorphisms were identified in coding and non-coding regions, including indels in the K3 and K15 genes and sequence inversions here reported for the first time. One such polymorphism in KSHV ORF46, specific to the KSHV K1 subtype E2, encoded a mutation in the leucine loop extension of the uracil DNA glycosylase that results in alteration of biochemical functions of this protein. This confirms that KSHV sequence variations can have functional consequences warranting further investigation. This study represents the largest and most diverse analysis of KSHV genome sequences to date among individuals with KAD and provides important new information on global KSHV genomics.

Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.

Engineering a solar formic acid/pentose (SFAP) pathway in Escherichia coli for lactic acid production

Microbial CO2 fixation into lactic acid (LA) is an important approach for low-carbon biomanufacturing. Engineering microbes to utilize CO2 and sugar as co-substrates can create efficient pathways through input of moderate reducing power to drive CO2 fixation into product. However, to achieve complete conservation of organic carbon, how to engineer the CO2-fixing modules compatible with native central metabolism and merge the processes for improving bioproduction of LA is a big challenge. In this study, we designed and constructed a solar formic acid/pentose (SFAP) pathway in Escherichia coli, which enabled CO2 fixation merging into sugar catabolism to produce LA. In the SFAP pathway, adequate reducing equivalents from formate oxidation drive glucose metabolism shifting from glycolysis to the pentose phosphate pathway. The Rubisco-based CO2 fixation and sequential reduction of C3 intermediates are conducted to produce LA stoichiometrically. CO2 fixation theoretically can bring a 20% increase of LA production compared with sole glucose feedstock. This SFAP pathway in the integration of photoelectrochemical cell and an engineered Escherichia coli opens an efficient way for fixing CO2 into value-added bioproducts.

Standardization of ALEX index for the ecological quality assessment under European Framework Directives in Mediterranean sensitive habitats affected by biological invasions

Abstract This paper aims at providing a standardized procedure to assess the ecological quality of Mediterranean marine habitats in relation to biological invasions by macroalgae through the employment of the ALEX index. Available data from previous ALEX applications were used to optimize the sampling design and to propose a standardized procedure. The procedure was applied to the main Mediterranean sensitive habitats, coralligenous reefs and meadows of the endemic seagrass Posidonia oceanica, at two sites in the north‐western Mediterranean Sea. A total of 73 native macroalgal taxa and four introduced taxa (the Chlorophyta Caulerpa cylindracea and the turf forming Rhodophyta Acrothamnion preissii, Womersleyella setacea and Asparagopsis spp.) were identified. Coralligenous reefs exhibited a higher abundance of both C. cylindracea and introduced turf species, while P. oceanica meadows were mostly affected by W. setacea and A. preissii. The application of ALEX for different years on coralligenous reefs highlighted the consistency of the index over time in the absence of further invasions. The use of ALEX index with other ecological quality indices showed similar patterns in both habitats. The concurrent assessment of the ecological quality of ecosystems by generic indices and the level of invasion through ALEX index may be a useful approach for answer to requirements of the European Directives and to plan even more complete and effective conservation programmes.

Deciphering the molecular mechanism and regulation of formaldehyde detoxification in Mycobacterium smegmatis.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

Macrocephaly and Digital Anomalies Expand the Phenotypic Spectrum of PGAP2 Variants in Hyperphosphatasia with Impaired Intellectual Development Syndrome 3 (HPMRS3)

Glycosylphosphatidylinositols (GPIs) anchor over 150 proteins as GPI-anchored proteins (GPI-APs) with crucial roles in diverse biological processes. The highly conserved biosynthesis of GPI-APs involves precise steps with at least 21 genes, categorized as PIG and PGAP genes. Pathogenic variants in these genes are linked to human diseases, highlighting the importance of each biosynthesis step. PGAP2 stands out among these genes due to its association with an expanded clinical spectrum of neurodevelopmental disorder (NDD) phenotypes with biallelic pathogenic variants. We present four patients from two families, one consanguineous and the other nonconsanguineous, each displaying distinct clinical presentations, including intellectual disability, hyperphosphatasia, hearing impairment, and epilepsy, as well as craniofacial and digital anomalies. Genetic analyses revealed homozygous and novel compound heterozygous missense variants in PGAP2 in four affected individuals, confirming the molecular diagnosis of hyperphosphatasia with impaired intellectual development syndrome 3 (HPMRS3). Importantly, the three amino acids affected by missense variants exhibit complete conservation in 10 vertebrate species, illuminating their crucial role in the gene’s functionality. Protein modeling provided additional evidence for the pathogenicity of the three substitutions, demonstrating their detrimental impact on protein folding and putative protein-protein interactions, ultimately leading to impaired protein function. The four patients in our study displayed common phenotypic features, such as brachydactyly, camptodactyly, and syndactyly, which have not been previously documented in individuals with PGAP2 variants. Notably, the occurrence of macrocephaly in two affected brothers from a consanguineous Pakistani family represents a novel finding. These previously unreported digital anomalies, along with macrocephaly and the identification of novel compound heterozygous variants, contribute to the expansion of the phenotypic and genotypic spectrum of HPMRS3 associated with PGAP2 variants.

Lessons from the deep: mechanisms behind diversification of eukaryotic protein complexes.

Genetic variation is the major mechanism behind adaptation and evolutionary change. As most proteins operate through interactions with other proteins, changes in protein complex composition and subunit sequence provide potentially new functions. Comparative genomics can reveal expansions, losses and sequence divergence within protein-coding genes, but in silico analysis cannot detect subunit substitutions or replacements of entire protein complexes. Insights into these fundamental evolutionary processes require broad and extensive comparative analyses, from both in silico and experimental evidence. Here, we combine data from both approaches and consider the gamut of possible protein complex compositional changes that arise during evolution, citing examples of complete conservation to partial and total replacement by functional analogues. We focus in part on complexes in trypanosomes as they represent one of the better studied non-animal/non-fungal lineages, but extend insights across the eukaryotes by extensive comparative genomic analysis. We argue that gene loss plays an important role in diversification of protein complexes and hence enhancement of eukaryotic diversity.

Evolution equations with complete irreversibility and energy conservation

This paper is concerned with the initial-boundary value problem for an evolutionary variational inequality complying with three intrinsic properties: complete irreversibility, unilateral equilibrium of an energy and an energy conservation law, which cannot generally be realized in dissipative systems such as standard gradient flows. Main results consist of well-posedness in a strong formulation, qualitative properties of strong solutions (i.e., comparison principle and the three properties mentioned above) and long-time dynamics of strong solutions (more precisely, convergence to an equilibrium). The well-posedness will be proved based on a minimizing movement scheme without parabolic regularization, which will also play a crucial role for proving qualitative and asymptotic properties of strong solutions. Moreover, the variational inequality under consideration will be characterized as a singular limit of some (generalized) gradient flows.

Investigation of a Listeria monocytogenes Chromosomal Immigration Control Region Reveals Diverse Restriction Modification Systems with Complete Sequence Type Conservation.

Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability.

Two new species of Pachira (Bombacoideae, Malvaceae) and typification of Pachira patinoi

Abstract We aimed to describe two new species endemic to Brazil - one from sandy habitats in northeastern Brazil (Pachira inaequalivalvis), mainly characterized by fruits with unequal valves, and the other from Amazonian rainforest (P. deflexifolia, having the downward-oriented leaflets) - and provide complete morphological descriptions, illustrations, comments, conservation status, and distribution maps. We also designate a lectotype and an epitype for Pachira patinoi.

Diversity of the Hydroxylamine Oxidoreductase (HAO) Gene and Its Enzyme Active Site in Agricultural Field Soils.

Nitrification is a key process in the biogeochemical nitrogen cycle and a major emission source of the greenhouse gas nitrous oxide (N2O). The periplasmic enzyme hydroxylamine oxidoreductase (HAO) is involved in the oxidation of hydroxylamine to nitric oxide in the second step of nitrification, producing N2O as a byproduct. Its three-dimensional structure demonstrates that slight differences in HAO active site residues have inhibitor effects. Therefore, a more detailed understanding of the diversity of HAO active site residues in soil microorganisms is important for the development of novel nitrification inhibitors using structure-guided drug design. However, this has not yet been examined. In the present study, we investigated hao gene diversity in beta-proteobacterial ammonia-oxidizing bacteria (β-AOB) and complete ammonia-oxidizing (comammox; Nitrospira spp.) bacteria in agricultural fields using a clone library ana-lysis. A total of 1,949 hao gene sequences revealed that hao gene diversity in β-AOB and comammox bacteria was affected by the fertilizer treatment and field type, respectively. Moreover, hao sequences showed the almost complete conservation of the six HAO active site residues in both β-AOB and comammox bacteria. The diversity of nitrifying bacteria showed similarity between hao and amoA genes. The nxrB amplicon sequence revealed the dominance of Nitrospira cluster II in tea field soils. The present study is the first to reveal hao gene diversity in agricultural soils, which will accelerate the efficient screening of HAO inhibitors and evaluations of their suppressive effects on nitrification in agricultural soils.

Chloroplast genome skimming of a potential agroforestry species Melia dubia. Cav and its comparative phylogenetic analysis with major Meliaceae members.

The online version contains supplementary material available at 10.1007/s13205-022-03447-1.

양주 회암사지 세계유산 등재를 위한 방향성 연구

As a result of more than a dozen excavations from the late 1990s to the present, the Yangju Hoeamsa Temple site has revealed that it is a representative zen temple site built with the support of the royal family from the late Goryeo to the early Joseon Dynasty. Since 2016, the Cultural Heritage Administration and Yangju City have been promoting research on the inscription of ‘Yangju Hoeamsa Temple Site’ as a World Heritage Site, and in January 2022, it was added to the World Heritage Tentative List.
 In order to be inscribed on the World Heritage List, it must meet the inscription criteria required by UNESCO, and must be recognized for its outstanding universal value combined with authenticity and integrity. In this process, it is also essential to establish a plan and system for the complete conservation and management of the property.
 The site of Hoeamsa Temple consists of nine serial heritages, including the site of the main building of the central ministry and the monuments of the high priests related to Hoeamsa. These elements are evidence that clearly shows the 200 years of transmission and development of the zen buddhism of Goryeo that led to the Joseon Dynasty, and are examples of concretely proving the spatial organization system of Seonjong temples, and can be considered to have outstanding universal value.
 In this article, regarding the direction to take for the inscription of the Hoeamsa Temple site as a World Heritage Site, the feasibility of component and zoning, the appropriateness of the inscription criteria, whether authenticity and integrity are met, the possibility of comparative studies of domestic and foreign heritage sites, conservation management plans, and other considerations matters were reviewed and opinions were presented.
 In the future, it is expected that the value of the Hoeamsa Temple site in Yangju will be in-depth comparatively studied in various aspects such as archaeology, architecture, and Buddhism, and the authorities concerned will actively support it so that the Hoeamsa Temple site can be inscribed as a UNESCO World Heritage Site.

TYPOLOGIA I ANALIZA SAKRALNEJ ARCHITEKTURY DREWNIANEJ NA TERENIE WOJEWÓDZ0TWA LUBUSKIEGO

The article presents research on the architecture and construction of wooden churches in the Lubuskie voivodeship, which form a large group of historical buildings. Some of them are monuments of high rank, both in terms of age and stylistic value, design solutions and equipment. The remaining ones are characterized by the common historical value of the wooden cultural heritage, which emphasizes the role of the local building tradition and the originality of the log and half-timbered architecture. The article presents seven types of wood and characterizes each of them. All researched monuments are under conservation protection. Most of them are renovated on an ongoing basis, and a few have undergone complete conservation, revalorization and revitalization in recent years.

Validation on the mean friction velocity of an atmospheric boundary layer flow reproduced by large-eddy simulation in terms of kinetic energy conservation

This study presents a validation of large-eddy simulation to reproduce normalized mean friction velocity in an atmospheric boundary layer flow in terms of kinetic energy conservation uncertainty. A primary finding of this study is that the normalised mean friction velocity of the atmospheric boundary layer is suggested to be insensitive to kinetic energy conservation errors. The present study approaches the sensitivity of the normalized friction velocity, which is one of the most fundamental statistics in the atmospheric boundary layer, to the uncertainty. The nearly complete conservation of kinetic energy in the present numerical framework is verified in an inviscid homogeneous isotropic fluctuation field. Then, the present analysis is applied to reproducing turbulent channel flows as direct numerical simulation and large-eddy simulation based on the previous studies before the present work analyses the present atmospheric boundary layer. The present analysis is then used to analyze the normalized friction velocity in the atmospheric boundary layer. The values of the friction velocity obtained in the previous study are in good agreement with that of the present study.

SUPPRESSOR OF GAMMA RESPONSE 1 plays rice-specific roles in DNA damage response and repair.

Land plants are constantly exposed to environmental stresses and have developed complicated defense systems, including DNA damage response (DDR) and DNA repair systems, to protect plant cells. In Arabidopsis (Arabidopsis thaliana), the transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) plays a key role in DDR. Here, we focus on DDR in rice (Oryza sativa)-thought to be a simpler system compared with Arabidopsis due to lack of induction of the endocycle even under DNA damage stress. Rice SOG1 (OsSOG1) and SOG1-like (OsSGL) were identified as putative AtSOG1 orthologs with complete or partial conservation of the serine-glutamine motifs involved in activation via phosphorylation. In addition to OsSOG1 or OsSGL knockout mutants, OsSOG1 nonphosphorylatable mutants (OsSOG1-7A) were generated by homologous recombination-mediated gene targeting. Based on the analysis of DNA damage susceptibility and the effect on the expression of DNA repair-related genes using these mutants, we have demonstrated that OsSOG1 plays a more important role than OsSGL in controlling DDR and DNA repair. OsSOG1-regulated target genes via CTT (N)7 AAG motifs reported previously as AtSOG1 recognition sites. The loss of transcription activity of OsSOG1-7A was not complete compared with OsSOG1-knockout mutants, raising the possibility that other phosphorylation sites might be involved in, or that phosphorylation might not be always required for, the activation of OsSOG1. Furthermore, our findings have highlighted differences in SOG1-mediated DDR between rice and Arabidopsis, especially regarding the transcriptional induction of meiosis-specific recombination-related genes and the response of cell cycle-related genes, revealing rice-specific DDR mechanisms.

Preparation of N-Aryl Amides by Epimerization-Free Umpolung Amide Synthesis.

Amide synthesis is one of the most widely practiced chemical reactions, owing to its use in drug development and peptide synthesis. Despite the importance of these applications, the attendant effort to eliminate waste associated with these protocols has met with limited success, and pernicious α-epimerization is most often minimized but not eliminated when targeting challenging amides (e.g., N-aryl amides). This effort has focused on what is essentially a single paradigm in amide formation wherein an electrophilic acyl donor reacts with a nucleophilic amine. Umpolung amide synthesis (UmAS) emerged from α-halo nitroalkane reactions with amines and has since been developed into a method for the synthesis of enantiopure amides using entirely catalytic, enantioselective synthesis. However, its inability to forge N-aryl amides has been a longstanding problem, one limiting its application more broadly in drug development where α-chiral N-aryl amides are increasingly common. We report here the reaction of α-fluoronitroalkanes and N-aryl hydroxyl amines for the direct synthesis of N-aryl amides using a simple Brønsted base as the promoter. No other activating agents are required, and experiments guided by mechanistic hypotheses outline a mechanism based on the UmAS paradigm and confirm that the N-aryl amide, not the N-aryl hydroxamic acid, is the direct product. Ultimately, select chiral α-amino-N-aryl amides were prepared with complete conservation of enantioenrichment, in contrast to a parallel demonstration of their ability to epimerize using the conventional amide synthesis alternative.

Primary diagnosis of energy efficiency in an integrated steel plant, based on intensive energy-saving methodology. Part 2

The energy efficiency of the integrated steel plant is studied basing on the intensive energy conservation methodology. The primary diagnostics of the energy efficiency of the integrated steel plant is carried out. The boundaries of the research object are established from iron ore deposits to finished cold-rolled steel sheet. The thermophysical heat absorption of materials and intermediates within the boundaries of a closed heat engineering complex has been estimated. The energy intensity of the cold-rolled steel sheet within the same boundaries has been calculated. Efficiency of production was estimated by comparing heat consumption and energy intensity. The complete intensive energy conservation reserve in the complex has been determined. Reserve structure is studied and directions of its implementation are discussed. It is shown that the most complete implementation of the energy-saving reserve is possible only with the transition of a new generation of steel engineering and technology.